Detection and quantification of nucleic acids are among the most important techniques in molecular biology and are rapidly evolving.
Basic classical techniques to analyze nucleic acids correspond to electrophoresis and probe hybridization that can be carried out in both the liquid and solid phase.
An important step in the development of nucleic acid manipulation techniques is the development of PCR (from the abbreviation Polymerase Chain Reaction).
The PCR technique (Saiki et al., Science, 230, 1350-1354 (1985), Mullis et al., North American patents U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 y U.S. Pat. No. 4,800,159), permits the exponential amplification of nucleic acids.
This amplification is achieved by repeated cycles of denaturisation of the nucleic acid studied, by heat, binding complementary primers to two opposing regions of the nucleic acid to be amplified, and extension of the nucleic acid by the action of a polymerase enzyme. The repetition of successive cycles of this process results in exponential amplification of the nucleic acid.
In the PCR technique, polymerases are used to obtain amplification of the nucleic acid studied.
DNA polymerases catalyze the synthesis of nucleic acids, and in spite of the fact that all of them can polymerize nucleic acids in the 5′-3′ region, there are differences among them in relation to the presence or absence of other characteristics, such as: exonuclease activity of the double helix, exonuclease activity of the single strand 3′-5′, exonuclease activity of the double strand 3′-5′, or reverse transcriptase activity.
The polymerases that present 3′-5′ exonuclease activity, perform DNA replication with much better accuracy since they proofread the errors in the replicated bases (Brutlag, D. And Kornberg, A. J. Biol. Chem. (1972) 247:241-248). When DNA polymerases with 3′-5′ exonuclease activity with proofreading activity are used in the replicator system, the DNA obtained includes a smaller proportion of base errors than replicas that do not use these types of enzymes (Chang, L. M. S., J. Biol.: Chem. (1977) 252:1873-1880.
Proofreading DNA polymerases with 3′-5′ exonuclease activity are well known. Documents such as patent with publication no. U.S. Pat. No. 5,500,363, and the patent with publication no. U.S. Pat. No. 5,352,778, include a description of how to obtain and produce a thermostable recombinant polymerase with 3′-5′ proofreading activity.
In the patent with publication number U.S. Pat. No. 6,489,150, the use of these types of DNA polymerases with 3′-5′ proofreading activity is described for the synthesis of nucleic acids.
In the patent with publication number WO0181631, a method is described to analyze in a nucleic acid variable sites in the presence of a polymerase with 3′-5′ exonuclease activity. This enzyme, in the case that one of the primers is not complementary to the target nucleic acid with the variable site, cuts the 3′ end of the primer, releasing the marker of this primer, after which the presence or absence of this marker is analyzed. By this method, one of the sequences present in the mixture is amplified and/or selectively labeled. However, the process does not generate a detectable signal, thus analysis of the presence of the marker requires an additional analysis of the products generated.
A PCR variant has been developed, real-time PCR, in which the amplification and detection processes are produced simultaneously, without requiring any further operation. Moreover, the amount of DNA synthesized at each moment can be measured by fluorescence detection during the amplification, since the fluorescence emitted during the reaction is proportional to the amount of DNA formed. Hence, the kinetics of the amplification reaction can be determined and known at any moment (Higuchi R, Fokler C, Dollinger G, Watson R. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Bio/Technology 1993; 11: 1026-30).
Currently, most equipment related to real-time PCR technology corresponds to the so-called thermocyclers that incorporate a fluorescence reader and are designed to measure, at any time, the fluorescence emitted in each of the test-tubes in which the amplification has been carried out.
The fluorescence detection systems currently most used in real-time PCR, are:                Fluorescent intercalating agents (such as SYBR Green)        Hydrolysis probes that use the 5′-3′ nuclease activity of the DNA polymerases (such as TaqMan probes and LNA probes)        Hairpin probes (such as those known as Molecular Beacons and Scorpion)        Hybridization probes (such as those known as FRET probes, TaqMan MGB probes and MGB Eclipse probes)        
The compound known with the brand name of SYBR Green and protected with the North American patent with publication number U.S. Pat. No. 5,436,134, is a much used fluorescent intercalating agent. This compound is a derivative of cyanine that binds to the double stranded nucleic acid, emitting a fluorescent signal that increases proportionally as the PCR product increases.
In a similar way as these intercalating agents bind to the nucleic acid products of the PCR, they can also bind to primer-dimers and to other non-specific products, and can have non-specific amplification signals resulting in an overestimation of the concentration of the target to be labeled.
Other kinds of fluorescent labeling systems correspond to hydrolysis probes, such as that with the brand name TaqMan, described in the patent of the invention with publication number U.S. Pat. No. 5,723,591. These probes are bound to a fluoride reporter at the 5′ end and a fluoride blocker or quencher at the 3′end. The Taqman probes, together with polymerases with 5′-3′ exonuclease activity, are used to monitor amplification of the nucleic acids. When both fluorides are bound to the probe, the reporter is diminished by the quencher and no signal is emitted. The probe is joined to the nucleic acid strand to be amplified, when this nucleic acid is replicated the 5′-3′ exonuclease activity of the DNA polymerase 5′-3′ exonuclease, frees the 5′ end of the probe where this is joined to the fluoride reporter, producing emission of a fluorescent signal.
Hairpin probes have inverted repeat sequences at their 5′ and 3′ ends, permitting a hairpin shaped structure to be formed owing to the complementarity of the two repeat inverted regions, in the absence of the target sequence. The internal sequence of the probe is complementary to the target sequence, so that in its presence, the hairpin structure opens, increasing the distance between the fluoride reporter and the fluoride quencher, so that the fluorescent signal is emitted.
Other known probes are the hybridization probes, the design of which involves the use of two specific oligonucleotide sequences as probes, each labeled with a different fluoride. The ends of the probe are complementary and usually the 3′ end of one of them is the donor. When this molecule is excited by a light source it transfers its energy to the 5′ end of the second probe, the acceptor molecule. The two probes are designed to hybridize in their specific targets so that both fluorides are in close proximity, so transfer of the resonance energy only occurs when both probes hybridize to the target, and are very close together.
There are other types of probe with a more limited and incipient use, such as the probes: “Resonsense”, “Light-up”, “HyBeacon”, “LUX”, “Yin-yang”, “Amplifluor” etc.
The present invention provides a new method to detect and quantify nucleic acid by the action of an enzyme with 3′-5′ nuclease activity.